1. Field of the Invention
The present invention relates to a microorganism strain capable of producing O-acetyl homoserine, an L-methionine precursor, in high yield. More particularly, the present invention relates to a microorganism strain in which an acetyl-CoA synthase gene and/or a pantothenate kinase gene refractory to feedback inhibition by CoA is introduced and enhanced to produce O-acetyl homoserine in high yield. Also, the present invention is concerned with a method of producing O-acetyl homoserine in high yield using the microorganism strain.
2. Description of the Related Art
Methionine, an essential amino acid for the body, finds a variety of applications in the food and medical industries, such as the use thereof as an additive in animal feed and foods and as a material for parenteral nutrient solutions and medicines. Methionine acts as a precursor for choline (lecithin) and creatine and is used as a material useful for the synthesis of cysteine and taurine. Together with cysteine, methionine is one of two sulfur-containing proteinogenic amino acids. S-Adenosyl methionine, derived from L-methionine, serves as a methyl donor in vivo and is involved in the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) is/are also found to prevent lipid accumulation in the liver and arteries and to alleviate depression, inflammation, liver diseases and muscle pain (Jeon B R et al., J Hepatol., 2001 March; 34(3): 395-401).
As summarized below, methionine and/or S-adenosyl-L-methionine has been thus far known to have the in vivo functions of:
1) suppressing lipid accumulation in arteries and in the liver, where lipid metabolism is mediated, and improving blood circulation in the brain, the heart and the kidneys (J Hepatol. Jeon B R et al., 2001 March; 34(3): 395-401).
2) promoting the digestion, detoxication and excretion of toxic substances and the excretion of heavy metals such as Pb.
3) acting as an antidepressant when methionine is administered in a daily dose of from 800 to 1,600 mg (Am J Clin Nutr. Mischoulon D. et al., 2002 November; 76(5): 1158S-61S)
4) improving liver functions against liver diseases (FASEB J. Mato J M., 2002 January; 16(1): 15-26), particularly, against alcohol-induced liver injury (Cochrane Database Syst Rev., Rambaldi A., 2001; (4): CD002235)
5) showing an anti-inflammation effect on osteoarthritis and promoting the healing of joints (ACP J Club. Sander O., 2003 January-February; 138(1): 21, J Fam Pract., Soeken K L et al., 2002 May; 51(5): 425-30).
6) acting as an essential nutrient to hair to prevent brittle hair and depilation (Audiol Neurootol., Lockwood D S et al., 2000 September-October; 5(5): 263-266).
For use in animal feed, foods and medicines, methionine can be synthesized chemically or biologically.
In the chemical synthesis, on the whole, methionine is produced through the hydrolysis of 5-(β-methylmercaptoethyl)-hydantoin. However, the synthesized methionine is disadvantageously present in a mixture of L- and D-forms which needs a difficult additional process to separate them from each other. In order to solve this problem, the present inventors developed a biological method for selectively synthesizing L-methionine, a chemical which a patent (WO 2008/103432) has already been applied for. The method, termed in brief “a two-step process”, comprises the fermentative production of an L-methionine precursor and the enzymatic conversion of the L-methionine precursor to L-methionine. The methionine precursor preferably includes O-acetylhomoserine and O-succinyl homoserine. The two-step process is evaluated on terms of having overcome the problems from which the conventional methods suffer, such as sulfide toxicity, feedback regulation in methionine synthesis by methionine and SAMe, and degradation of intermediates by cystathionine gamma synthase, O-succinylhomoserine sulfhydrylase and O-acetylhomoserine sulfhydrylase. Also, compared to the conventional chemical synthesis method of producing DL-methionine, the two-step process has the advantage of being selective for L-methionine only, with the concomitant production of organic acids, such as succinic acid and acetic acid as useful by-products.
Found as an intermediate in the biosynthesis pathway of methionine, O-acetyl-homoserine is used as a precursor for the production of methionine (WO 2008/013432). O-acetyl-homoserine is synthesized from L-homoserine and acetyl-CoA with the aid of O-acetyl transferase as shown in the following formula:    L-Homoserine+Acetyl-CoA O→Acetyl-Homoserine.
In the U.S. patent application Ser. No. 12/062,835 of the present assignee are disclosed a microorganism strain in which thrA and metX genes are introduced to improve the biosynthesis of L-homoserine and O-acetyl-homoserine, respectively, and a method for producing O-acetyl homoserine at high yield using the same. In this context, the present inventors conceived that the enrichment of acetyl-CoA, one of the two substrates used in the biosynthesis of O-acetyl-homoserine, would increase the production yield of O-acetyl homoserine.
In E. coli, acetyl-CoA is, for the most part, synthesized from pyruvate. However, if excess glucose is present, acetyl-CoA may be produced from the acetic acid accumulated in the medium. The synthesis of acetyl-CoA from acetic acid may take advantage of two different biosynthesis pathways. In the one acetyl-CoA biosynthesis pathway, an acetyladenylate (AcAMP) intermediate is found through which acetyl-CoA synthase (ACS) activates acetate to acetyl-CoA. In the other pathway, acetic acid is converted through acetyl phosphate to acetyl-CoA as a result of the consecutive reactions catalyzed by acetate kinase (ACK) and phosphotransacetylase (PTA) (JOURNAL OF BACTERIOLOGY, May 1995, p. 2878-2886). Having a high affinity for acetate, acetyl-CoA synthase, which plays a pivotal role in the biosynthesis pathway mediated thereby, can activate acetate even at a low intracellular or extracellular level to acetyl-CoA. In contrast, the ACK-PTA-mediated acetyl-CoA biosynthesis pathway is operated only at a high acetate level, which may result from the fermentation of mixed acids, because the enzymes have a low affinity for acetate (J. Gen. Microbiol. 102:327-336.).
As for the acetyl-CoA synthase, its expression is inhibited at the transcription level by catabolite repression control until the exponential growth due to the CRP-binding site located upstream of the promoter and since then, its expression increases when the cell enters the stationary phase (Mol. Microbiol. 2004 January; 51(1):241-54.).
Thus, the acetate accumulated in the middle phase of fermentation can be activated to acetyl-CoA through the ACK-PTA pathway. However, if there is a low level of acetate, acetyl-CoA is converted to acetate because the ACK-PTA pathway is reversible. That is, the depletion of acetyl-CoA may occur during the ACK-PTA pathway, showing a negative effect on the synthesis of O-acetyl homoserine.
Accordingly, in order to utilize the acetate produced during fermentation, efforts are made to increase the activity of acetyl-CoA synthase. The increase of acetyl-CoA synthase activity allows the acetate accumulated in the media to be rapidly converted into acetyl-CoA, resulting in an improvement in the production of O-acetyl homoserine.
Coenzyme A (CoA), used as a substrate, together with acetate, in the biosynthesis of acetyl-CoA, is a representative acyl group carrier within cells. Coenzyme A is synthesized in a series of processes from pantothenate with enzymatic catalysis for each process as follows. First, pantothenate kinase (CoaA) activates pantothenate (Vitamin B5) to 4′-phosphopantothenate to which a cysteine is then added to form 4′-phosphopantothenoyl-L-cysteine, followed by decarboxylation to 4′-phosphopantetheine by the cooperation of P-PanCys synthase/P-PanCys decarboxylase (coaBC). Subsequently, 4′-phosphopantetheine is adenylylated to form dephospho-CoA by the enzyme phosphopantetheine (P-PanSH) adenylyltransferase (coaD). Finally, dephospho-CoA is phosphorylated using ATP to coenzyme A by the enzyme dephosphocoenzyme A (deP-CoA) kinase (coaE).
Generally, CoA is a cofactor for a multitude of metabolic reactions as well as many synthetic reactions within cells. For this reason, its pool is maintained at a constant level by regulation mechanisms. The primary key player in regulating the intracellular CoA pool is pantothenate kinase, which catalyzes the first committed step and is the rate-controlling enzyme in CoA biosynthesis. The regulation of pantothenate kinase activity by feedback inhibition is the critical factor controlling the intracellular CoA concentration (J Biol. Chem. 1994 Oct. 28; 269(43):27051-8). However, the constant CoA level maintained within cells may be a barrier to the effective production of O-acetyl homoserine through acetyl-CoA. It is reported that the substitution of arginine R at position 106 with alanine A alters pantothenate kinase from being sensitive to feedback inhibition by CoA to being refractory thereto (JOURNAL OF BACTERIOLOGY, 185, June 2003, p. 3410-3415). The wild-type protein was found to retain only about 20% of the catalytic activity in the presence of 40 mM CoA whereas no decreases were detected in the catalytic activity of the R106A mutant protein at the same concentration of CoA. Further, a mutant strain which expressed the mutant protein was found to have significantly higher intracellular levels of CoA, compared to the wild-type.
Leading to the present invention, intensive and thorough research, conducted by the present inventors, resulted in the finding that the introduction and enhancement of either or both of (a) the pantothenate kinase gene refractory to feedback inhibition by CoA and (b) the O-acetyl CoA synthase gene results in a significant improvement in the productivity of O-acetyl homoserine.